We have established a system to investigate envelope-mediated crosslinking of CD4+ T lymphocytes in vitro. With this system we have addressed the potential of envelope proteins to mediate apoptosis in peripheral blood mononuclear cells (PBMCs). To specifically address the role of envelope binding to CD4 receptors in the absence of other phenomenon associated with viral infection, we have expressed, purified, and characterized recombinant envelope proteins from a mammalian expression system. We have produced two classes of recombinant proteins, those comprised of the SU protein gp120 and a second comprised of the SU/TM protein complex gp120/41. We have shown that the gp120 protein exists in solution as a monomer whereas the gp120/41 complex forms a multimeric complex of at least three gp120/41 subunits. We have utilized several assays evaluating cell proliferation, anergy and apoptosis to assess the activity of these proteins on whole PBMCs. We find that multimeric gp120/41 protein complexes induce apoptosis and inhibit cell proliferation in various subpopulations of PBMCs while monomeric gp120 does not. To the extent that apoptosis is induced by multimeric envelope proteins mediated crosslinking of CD4 receptors, we have begun to measure the stability of various envelope multimers of HIV-1 and SIV. The isolates being studied represent different clades of HIV as well as viruses with distinct tropisms e.g. macrophage/monocyte versus T-cell tropic isolates. Our initial observations indicate that multimers of SIV are far more stable than multimers of HIV. We plan to further characterize the basis for these differences in stability and to attempt to assess their impact on apoptosis and anergy of whole PBMC cultures. Additionally, we will attempt to identify subsets of lymphocytes that undergo apoptosis and establish the mechanism by which CD4 negative lymphocytes in PBMC cultures undergo apoptosis after treatment with envelope protein.